The cellular response to hypoxia is massive. Hypoxia alters the expression of hundreds of genes, but specific findings seem to conflict, with some genes upregulated in certain experiments but downregulated in others. Gaining a clear understanding of the transcriptional response to low-oxygen conditions is critical to understanding the pathways involved in the response to hypoxia. In the January issue of G3, Bendjilali et al. take a more detailed look at the effects of hypoxia in the budding yeast Saccharomyces cerevisiae, focusing on how cells react to the first shock of hypoxia and how this response changes as cells adapt over time.

A key component of the team’s approach was a technique called RNA-seq, which has many advantages over previously used methods, such as microarrays. Using next-generation sequencing to determine the relative abundance of RNAs, RNA-seq can detect and quantify transcripts of markedly low or high abundance, and the results are very reproducible. The researchers also collected data at many points in time before and after depriving the yeast of oxygen, which allowed them to detect transient changes in gene expression and to monitor gene expression kinetics. This enabled them to reveal the order in which each group of genes changes. Importantly, they were able to distinguish between the cells’ immediate, short-term response to hypoxia and the transition to a stable hypoxic state.

Using this method, Bendjilali et al. found that there are large changes in the transcription of many genes within the first two hours of growth in a low-oxygen environment. After that point, the changes level out, suggesting that a stable state for survival in such conditions is reached. The analysis identified 816 oxygen-regulated genes, a large portion of which had either not been previously associated with hypoxia or had been inconsistently identified by prior studies. Many of the newly-found oxygen-regulated genes were expressed at low levels, which may explain why they were not detected well by microarrays.

Many of the hypoxia-regulated genes were involved in predicted pathways, such as lipid metabolism and cellular respiration. However, some of the genes were involved in processes not previously linked to hypoxia. For example, some genes responsible for B vitamin metabolism were downregulated in response to hypoxia, possibly because B vitamin biosynthesis involves oxygen-dependent and oxygen-sensitive steps, and some B vitamins are important for anaerobic respiration.

Even more unexpectedly, some genes involved in the response to oxidative stress were upregulated during the hypoxic response. Digging deeper, the researchers found that of nine genes involved in dealing with oxidative stress, seven were upregulated while two were downregulated. This may be because while oxidative stress is generally reduced in low-oxygen states, the types of reactive oxygen species generated during hypoxia are different from those produced under normal conditions. These results suggest that the entire pathway is fine-tuned rather than simply up- or downregulated in response to hypoxia.

Interestingly, Bendjilali et al. also found evidence that the hypoxic response is quite distinct from the environmental stress response (ESR), a generalized reaction to a wide range of stressors, such as DNA damage and oxidative stress. The ESR genes that did change in response to hypoxia generally exhibited a transient shift in transcription (over 30 minutes or so) before returning to baseline.

These results suggest that hypoxia causes mild stress, but that the hypoxic response leads to a distinct state characterized by a stable adaptation to low-oxygen conditions. Hypoxia is a unique condition that elicits a massive transcriptional response, and further research will be required to understand how each of the many pathways uncovered by Bendjilali et al. are optimized for life in low oxygen.